TROYSOL LAC

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested in three different in vitro genetic toxicity studies, both with and without metabolic activation. The test article did not induce gene mutations by base pair changes or frameshifts in an Ames test. The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay and thus is not considered to be mutagenic in mammalian cells. Additionally, the test item did not induce structural chromosome aberrations as determined by the in vitro chromosome aberration test in V79 cells. Overall, the test item is not consiered to be mutagenic in bacterial and mammalian cells as well as not clastogenic in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 27, 1998 - July 20, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Directive 92/69

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver S9-mix from rats induced with ß-naphthoflavone

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Solvent: DMSO
Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test article can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Rationale for test conditions:

According to guideline

Evaluation criteria:

A test article is considered positive if either a reproducible duse related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

A test article producing neither a reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice on TA 1535 and TA 1537.

Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether ihe highest dose induced the criteria described above or not.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occured.

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100 eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Since no cytoxicity occured up to the highest concentration (5000 µg/plate), this concentration was chosen as maximum concentration for the main experiment. Applying an adequate spacing factor the following concentrations were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data:
- S9 mix: TA1535: 68-814; TA1537: 42-191; TA98: 86-450; TA100: 460-910; TA102: 751-1395

- Negative (untreated) historical control data:
- S9 mix: TA1535: 9-29; TA1537: 5-28; TA98: 15-57; TA100: 77-189; TA102: 121-293

- Solvent historical control data:
- S9 mix: TA1535: 9-25; TA1537: 4-28; TA98: 14-58; TA100: 77-225; TA102: 121-295

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study according to OECD Guideline 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and III) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The experiments were performed with and without liver microsomal activation. In experiment II, no unambiguous positive response was reached in strain TA 100 with S9 mix (factor 2.1) and in strain TA 102 with (factor 2.2) and without S9 mix (factor 2.4) Therefore, these result were dismissed and an additional experiment was performed with strains TA 100 with S9 mix and TA 102 with and without S9 mix. The results of this additional experiment are reported as experiment II. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

33, 100, 333, 1000, 2500, and 5000 µg/plate.

No relevant toxic effects, evident as a reduction in the number of revenants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 08, 1998 - November 11, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: supplied by Laboratory for Mutagenicity Testing, LMP, Technical Univeisity Darmstadt.
- Suitability of cells: Especially the high proliferation rate (doubling time of clone V79/T5 in stock cultures) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 h
- Methods for maintenance in cell culture: Large stocks of the cell line were stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in experiments.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum, humidified atmosphere with 4.5% CO2 at 37°C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver induced by ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
with and without S9 mix; 4 hours exposure and 18 h preparation interval
10, 20, and 40 µg/mL

Experiment II:
without S9 mix; 18 hours exposure and 18 h preparation interval
20, 40, and 80 µg/mL

without S9 mix; 28 hours expsoure and 28 hours preparation interval
80 µg/mL

with S9 mix; 4 hours exposure and 28 hours preparation interval
20, 40, and 80 µg/mL

Vehicle / solvent:

Solvent: Ethylene glycol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1x10^4 - 6x10^4

DURATION
- Exposure duration: Experiment I (with and without S9 mix): 4 hours; Experiment II (without S9 mix): 18 and 28 hours, respectively; Experiment II (with S9 mix): 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours, respectively

SPINDLE INHIBITOR: Colcemid (0.2 µg/mL)

STAIN: GIEMSA solution

NUMBER OF REPLICATIONS: Two repilcations in each experimental group

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 2 h after treatment with colcemid, cells were treated with 0.4% KCl for 20 min at 37°C. Afterwards, cells were fixed with 3 + 1 methanol + glacial acetic acid. After preparation the cells were stained with Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 well spread metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell number

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was determined of each test group in a sample of 500 cells per culture.

Rationale for test conditions:

According to Guideline (version 21 July 1997)

Evaluation criteria:

A test article is classified as non-mutagenic if
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps), no significant increase ofthe number of structural chromosome aberrations are observed

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0-4 0 % aberrant cells exclusive gaps],
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Both biological and statistical significance should be considered together.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 40 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 80 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: Precipitation of the test article in culture medium was observed after treatment with 312.5 µg/mL and above. Precipitation did not influence the study results as the maximum tested concentration was 80.0 µg/mL.

RANGE-FINDING/SCREENING STUDIES: Test article concentrations between 39.06 and 5000 µg/mL (with and without S9-mix)| were chosen for the evaluation of cytotoxicity. In the absence and the presence of S9 mix, strong toxic effects were observed starting at the lowest applied concentration of 39 µg/mL. Therefore, 40 µg/mL was chosen as top concentration in experiment I in the absence and presence of S9 mix. The concentrations for continuous treatment in experiment II the absence of S9 mix were chosen considering the data of the pre-test on toxicity and the first experiment. Since the toxicity data of experiment I showed no strong response, 160 µg/mL was chosen as top treatment concentration in experiment II. In experiment II, the cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible due to strong test article induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality). Thus, a maximum concentration of 80 µg/mL was chosen.

Conclusions:

The test item did not induce structural chromosome aberrations as determined by the in vitro chromosome aberration test in V79 cells. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article, dissolved in ethylene glycol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. II) after start of treatment with the test article. In experiment I, the exposure period was 4 h with and without metabolic activation. In experiment II the exposure period was 4 h with S9 mix and 18 h and 28 h without S9 mix.

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. The highest applied concentration in the pre-test (5000 µg/mL) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 39.06 and 5000 µg/mL (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. Strong toxicity was detected after treatment with 39.06 µg/mL and above in the absence and presence of S9 mix.

 

Therefore, the following concentrations were chosen:

Experiment I:

with and without S9 mix; 4 hours exposure and 18 h preparation interval

10, 20, and 40 µg/mL

 

Experiment II:

without S9 mix; 18 hours exposure and 18 h preparation interval

20, 40, and 80 µg/mL

without S9 mix; 28 hours expsoure and 28 hours preparation interval

80 µg/mL

with S9 mix; 4 hours exposure and 28 hours preparation interval

20, 40, and 80 µg/mL

 

In this study no influence of the test article on the pH value or osmolarity was observed.

In the absence of S9 mix in experiment II a slightly reduced mitotic index was observed after continuous treatment with 80 µg/mL at the 28 h interval. In addition reduced cell numbers were observed at 40 µg/mL (18 h interval) after 4 h treatment. In the presence of S9 mix, toxic effects were observed after treatment with 40 pg/ml (exp. I) and 80 µg/mL at the 18 h interval (exp. II).

In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed.

In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0 05) in cells with structural chromosome aberrations.

 

The test item did not induce structural chromosome aberrations as determined by the in vitro chromosome aberration test in V79 cells. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration test.

 

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 02, 1998 - September 07, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

additional experiment 1b in presence of metabolic activation since insuffficient toxicity was achieved in the first experiment.

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
- Suitability of cells: The cell line has successfully been used in in vitro experiments for many years.
- Doubling time: 10 -12 hours (cloning efficiency of untreated cells more than 50%)
- Methods for maintenance in cell culture: Large stocks of the cleansed L5178Y cell line are stored liquid nitrogen allowing the repeated use of the same cell culture batch.
- Modal number of chromosomes: near diploid (40 +/- 2) chromosome number

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 complete culture medium, humidified atmosphere with 4.5% CO2 and 95.5% air, 37°C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes/no
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver induced by ß-Naphthoflavone

Test concentrations with justification for top dose:

Experiment I with S9 mix: 3.8*; 7.5; 15; 30; 40; 60 µg/mL
Experiment I without S9 mix: 3.8*; 7.5; 15; 30; 40; 60 µg/mL
Experiment Ib with S9 mix: 70; 80 µg/mL
Experiment II without S9 mix: 3.8*; 7.5; 15; 30; 40; 60 µg/mL

Although the recommended range of 10 - 20 % was not actual reached the results are acceptable since the spacing of concentrations was below a factor of 2.

Vehicle / solvent:

- Solvent: Ethylene glycol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethylene glycol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 10E+7 cells/flask

DURATION
- Exposure duration: 4 hours (experiment I), 24 hours (experiment II)
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 – 15 days

SELECTION AGENT (mutation assays): 100 pg/mL TFT

NUMBER OF REPLICATIONS: Three independent experiments, each using two parallel cultures.

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG), relative total growth (RTG), cloning efficiency

Rationale for test conditions:

According to Guideline (version 21 July 1997)

Evaluation criteria:

A test article is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.

A test article producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A significant response is described as follows:

- The test article is classified as mutagenic if it reproducibly induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.
- The test article is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
- However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
- Results of test groups are rejected if either, the relative total growth or relative suspension growth is less than 10 % of the solvent control or the cloning efficiency after the expression period is less than 20 %.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
According to the toxicity pre-experiments the concentration range was selected. In the first pre-experiments strong toxic effects were observed at 39.1 µg/mL and above with and without S9 mix. The second pre-experiment showed no relevant toxic effects up to 40 µg/mL. Strong toxic effects occurred at 60 µg/mL in the absence of metabolic activation in experiment I and at 70 and 80 µg/mL in the presence of metabolic activation in experiment lb. In the second experiment no relevant toxic effects were observed up to 60 µg/mL.

Conclusions:

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5J78Y in the presence and absence of metabolic activation.
Therefore, the test item is considered to be non mutagenic in this mouse Lymphoma thymidine kinase locus assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures. The first main experiment was performed with and without liver microsomal activation .

 Since the toxic range was not quite reached with the doses used in the first experiment in the presence of metabolic activation, an additional experiment (experiment Ib) was required to provide data in the toxic range of the test item with metabolic activation. The second experiment was performed in the absence of metabolic activation only with a treatment period of 24 hours. The test article was tested at the following concentrations:

 

Experiment I:

without S9 mix: 3 8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

with S9 mix 3 8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

Experiment Ib:

with S9 mix 70.0 and 80.0 µg/mL

Experiment II:

without S9 mix 3.8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

* culture was not continued since only a minimum of four concentrations is required by the guidelines

 

According to the toxicity pre-experiments the concentration range was selected. In the first pre-experiments strong toxic effects were observed at 39.1 µg/mL and above with and without S9 mix. The second pre-experiment showed no relevant toxic effects up to 40 µg/mL. Strong toxic effects occurred at 60 µg/mL in the absence of metabolic activation in experiment I and at 70 and 80 µg/mL in the presence of metabolic activation in experiment lb. In the second experiment no relevant toxic effects were observed up to 60 µg/mL.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed, neither in the presence nor in the absence of metabolic activation. No relevant shift in the ratio of small versus large colonies occurred up to the maximal concentration tested.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5J78Y in the presence and absence of metabolic activation.

Therefore, the test item is considered to be non-mutagenic in this mouse Lymphoma thymidine kinase locus assay.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

This study according to OECD Guideline 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and III) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The experiments were performed with and without liver microsomal activation. In experiment II, no unambiguous positive response was reached in strain TA 100 with S9 mix (factor 2.1) and in strain TA 102 with (factor 2.2) and without S9 mix (factor 2.4) Therefore, these result were dismissed and an additional experiment was performed with strains TA 100 with S9 mix and TA 102 with and without S9 mix. The results of this additional experiment are reported as experiment II. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

33, 100, 333, 1000, 2500, and 5000 µg/plate.

No relevant toxic effects, evident as a reduction in the number of revenants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

In vitro mammalian cell gene mutation test

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures. The first main experiment was performed with and without liver microsomal activation .

 Since the toxic range was not quite reached with the doses used in the first experiment in the presence of metabolic activation, an additional experiment (experiment Ib) was required to provide data in the toxic range of the test item with metabolic activation. The second experiment was performed in the absence of metabolic activation only with a treatment period of 24 hours. The test article was tested at the following concentrations:

 

Experiment I:

without S9 mix: 3 8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

with S9 mix 3 8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

Experiment Ib:

with S9 mix 70.0 and 80.0 µg/mL

Experiment II:

without S9 mix 3.8*, 7 5, 15.0, 30.0, 40.0, and 60.0 µg/mL

* culture was not continued since only a minimum of four concentrations is required by the guidelines

 

According to the toxicity pre-experiments the concentration range was selected. In the first pre-experiments strong toxic effects were observed at 39.1 µg/mL and above with and without S9 mix. The second pre-experiment showed no relevant toxic effects up to 40 µg/mL. Strong toxic effects occurred at 60 µg/mL in the absence of metabolic activation in experiment I and at 70 and 80 µg/mL in the presence of metabolic activation in experiment lb. In the second experiment no relevant toxic effects were observed up to 60 µg/mL.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed, neither in the presence nor in the absence of metabolic activation. No relevant shift in the ratio of small versus large colonies occurred up to the maximal concentration tested.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5J78Y in the presence and absence of metabolic activation.

Therefore, the test item is considered to be non-mutagenic in this mouse Lymphoma thymidine kinase locus assay.

In vitro mammalian chromosome aberration test

The test article, dissolved in ethylene glycol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. II) after start of treatment with the test article. In experiment I, the exposure period was 4 h with and without metabolic activation. In experiment II the exposure period was 4 h with S9 mix and 18 h and 28 h without S9 mix.

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. The highest applied concentration in the pre-test (5000 µg/mL) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 39.06 and 5000 µg/mL (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. Strong toxicity was detected after treatment with 39.06 µg/mL and above in the absence and presence of S9 mix.

 

Therefore, the following concentrations were chosen:

Experiment I:

with and without S9 mix; 4 hours exposure and 18 h preparation interval

10, 20, and 40 µg/mL

 

Experiment II:

without S9 mix; 18 hours exposure and 18 h preparation interval

20, 40, and 80 µg/mL

without S9 mix; 28 hours expsoure and 28 hours preparation interval

80 µg/mL

with S9 mix; 4 hours exposure and 28 hours preparation interval

20, 40, and 80 µg/mL

 

In this study no influence of the test article on the pH value or osmolarity was observed.

In the absence of S9 mix in experiment II a slightly reduced mitotic index was observed after continuous treatment with 80 µg/mL at the 28 h interval. In addition reduced cell numbers were observed at 40 µg/mL (18 h interval) after 4 h treatment. In the presence of S9 mix, toxic effects were observed after treatment with 40 pg/ml (exp. I) and 80 µg/mL at the 18 h interval (exp. II).

In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed.

In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0 05) in cells with structural chromosome aberrations.

 

The test item did not induce structural chromosome aberrations as determined by the in vitro chromosome aberration test in V79 cells. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration test.

 

 

 

 

Justification for classification or non-classification

Based on results of three different in vitro genetic toxicity studies, the test item was not classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).